The present invention relates to conjugates of diagnostic or therapeutic principles, such as drugs, toxins, chelators, boron compounds and detectable labels, to an antibody, in which the diagnostic or therapeutic principle is first loaded onto a polymer carrier such as an aminodextran or a polypeptide of at least 50 amino acids in length, and this intermediate is in turn site-specifically conjugated to a targeting antibody such as an antitumor antibody. The resultant conjugate targets the diagnostic or therapeutic principle to a target tissue or organ where the diagnostic or therapeutic effect is realized.
Conjugation of cytotoxic drugs to antibodies to achieve a targeted therapeutic result is known. In particular, it is known that methotrexate (MTX) can be conjugated to antibodies and some selective cytotoxicity has been observed. It is desirable to enhance the selectivity and cytotoxicity of such conjugates by increasing the antibody loading of the cytotoxic drug. However, multiple conjugation of individual drug molecules to an antibody eventually reduces its immunoreactivity, the effect being observed when more than about ten drug molecules are loaded.
It has been suggested that the drug be conjugated to an intermediate polymeric carrier, which in turn would be conjugated to antibody. This has the advantage that larger numbers of drug molecules can be attached to the antibody at fewer sites on the antibody itself, so that immunoreactivity is not as seriously compromised.
One approach has been to attach MTX to bovine serum albumin (BSA) and then randomly link the intermediate to antibody, as reported by Garrett et al., Int. J. Cancer, 31:661-670, 1983. These authors were able to attach about 37 MTX molecules to BSA (average molecular weight of 70,000) but the resultant antibody conjugate had an immunoreactivity of only about 28% of that of the intact antibody.
Use of polylysine as a polymer carrier was reported by Ryser et al., Proc. Natl. Acad. Sci. USA, 75:3867-3870, 1978. These authors found that only about 13 MTX per carrier could be loaded and immunoreactivity was poor. In addition, the high amine content of the polymer, largely in the form of charged ammonium groups, caused the conjugate to stick to normal cells and vitiated the selectivity of the cytotoxic effect.
Rowland, U.S. Pat. No. 4,046,722, discloses an antibody conjugate wherein a plurality of molecules of a cytotoxic agent are covalently bound to a polymer carrier of molecular weight 5000-500,000, and the loaded carrier is covalently bound to an antibody by a random attachment to pendant amine or carboxyl groups. The covalent attachment is effected either by direct condensation to form amide bonds between amine groups on one and carboxyl groups on the other component of the conjugate, or by glutaraldehyde linkage of amine groups on the carrier to amine groups on the antibody. Again, this has the disadvantages of loss of immunoreactivity of the antibody and some risk of crosslinking of the antibody and/or the carrier. Ghose et al., J Natl Cancer Inst, 61:657-676, 1978, disclose other antibody-linked cytotoxic agents useful for cancer therapy, but again, covalent attachment is not to the oxidized carbohydrate portion of the antibody. These references show that it is well known in the art to prepare drug-loaded polymer carriers, but that their mode of attachment to antibodies in the past has been random, through pendant amine or carboxyl groups on the antibody.
Chelating groups for radiometals and/or metal ions which can act as magnetic resonance enhancing agents have been covalently bound to antibodies by a variety of methods, most involving random attachment to pendant amine, carboxyl, sulfhydryl, or phenyl groups on the polypeptide chain. Toxins and boron addends have also been linked to antibodies by various methods for targeted therapy, the boron groups being activated by thermal neutron irradiation once they have been localized at the site of a tumor or other pathological lesion by the antibody targeting vehicle to which they are bound. Detectable labels, such as enzymes, DNA segments, fluorescent compounds and the like, have been bound to antibodies for use in assays, again by random coupling to pendant groups.
In an attempt to avoid the undesirable effects of random coupling to antibody and crosslinking, McKearn, et al., in European Patent Application No. 88,695, published on Sept. 14, 1983, disclose a method for preparing antibody conjugates which involves oxidizing the carbohydrate portion of the antibody and linking compounds with free amine groups to the resultant carbonyls (aldehyde and/or ketone groups) by Schiff base formation and optional reductive stabilization. This reference discloses site-specific attachment of a variety of compounds, such as chelators, drugs, toxins, detectable labels and the like, to the oxidized carbohydrate portion of an antibody. Short peptide linkers are disclosed to provide spacers between these compounds and the antibody, either to make the linkage more readily cleaved or resistant to cleavage at the target site. Attachment of the oxidized carbohydrate to a polymer such as an aminodextran is disclosed, but only in the context of linking the antibody to an insoluble support such as a polymer coated bead, plate or tube, such as would be used in an immunoassay. There is no suggestion in this reference to covalently bind a polymer carrier loaded with functional molecules such as drugs, chelators or the like, to the oxidized carbohydrate portion of an antibody to prepare a soluble conjugate for use as a diagnostic or therapeutic agent.
A need therefore continues to exist for an antibody conjugate of a diagnostic or therapeutic principle, such as a drug, toxin, chelator, boron compound or detectable label, that combines high loading with minimal decrease in immunoreactivity for selective targeting of the diagnostic or therapeutic principle to a target tissue or organ, or for highly efficient and sensitive immunoassay or immunohistological applications.